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An Efficient Genetic Screen in Drosophila to Identify Nuclear

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An Efficient Genetic Screen in Drosophila to Identify Nuclear Copyright Ó 2006 by the Genetics Society of America DOI: 10.1534/genetics.106.061705 Note An Efficient Genetic Screen in Drosophila to Identify Nuclear-Encoded Genes With Mitochondrial Function T. S. Vivian Liao,*,1 Gerald B. Call,†,1 Preeta Guptan,† Albert Cespedes,† Jamie Marshall,† Kevin Yackle,† Edward Owusu-Ansah,† Sudip Mandal,† Q. Angela Fang,† Gelsey L. Goodstein,† William Kim† and Utpal Banerjee*,†,‡,2 *Molecular Biology Institute, †Department of Molecular, Cell and Developmental Biology and ‡Department of Biological Chemistry, University of California, Los Angeles, California 90095 Manuscript received June 8, 2006 Accepted for publication July 6, 2006 ABSTRACT We conducted a screen for glossy-eye flies that fail to incorporate BrdU in the third larval instar eye disc but exhibit normal neuronal differentiation and isolated 23 complementation groups of mutants. These same phenotypes were previously seen in mutants for cytochrome c oxidase subunit Va. We have molecularly characterized six complementation groups and, surprisingly, each encodes a mitochondrial protein. Therefore, we believe our screen to be an efficient method for identifying genes with mitochondrial function. ITOCHONDRIAL function is essential for a (Flores et al. 1998). However, in the case of tend, such M number of important cellular processes, such defects are caused by the lack of a sufficient number of as the generation of ATP (reviewed in Ackerman and precursor cells from which cone and pigment cells arise Tzagoloff 2005), apoptosis (reviewed in Newmeyer (Mandal et al. 2005). and Ferguson-Miller 2003), and the regulation of The events that give rise to the tend adult eye phe- aging (reviewed in Ohta 2003). Recently, work from notype occur in larval eye development. The larval our laboratory has shown that the metabolic status of a Drosophila eye imaginal disc undergoes two distinct cell, controlled by mitochondrial function, also regu- phases of proliferation (Wolff and Ready 1991). andal lates the G1–S checkpoint in mitosis (M et al. During the early larval stages prior to differentiation, 2005). This is evident in tenured (tend) mutants, which all cells divide frequently and asynchronously, appar- contain a null mutation in the gene encoding cyto- ently without regulation. In the third larval instar, the chrome c oxidase subunit Va of complex IV of the morphogenetic furrow sweeps across the eye disc to mitochondrial electron transport chain. This mutation pattern the cells (Ready et al. 1976). Within the mor- causes a reduction in ATP generated in mutant cells to phogenetic furrow, cells arrest in G1 and, following the 40% of wild-type levels, triggering the activation of passage of the furrow, either differentiate into neuronal AMPK and p53, which leads to the eventual down- photoreceptors or reenter the cell cycle and undergo a regulation of cyclin E. A G1–S mitotic checkpoint is terminal round of division termed the second mitotic then enforced, preventing cells from reentering the wave (SMW). This generates the precursors for cone cell cycle (Mandal et al. 2005). and pigment cell lineages (Wolff and Ready 1991). Adult tend mutants have glossy eyes lacking lens Misregulation of the SMW will lead to a loss of these material, normally secreted by cone and pigment cells accessory cell types. In the third larval instar, cells of the eye. In earlier studies, the lens-depleted glossy-eye mutant for tend anterior to the furrow slow down in cell phenotype was described in lozenge mutants, which are cycle progression, while those posterior to the furrow defective in cone and pigment cell specification fail to cross the G1–S checkpoint due to impaired ATP production (Mandal et al. 2005). Therefore, in addi- tion to undergoing fewer rounds of mitosis prior to the 1 These authors contributed equally to this work. passage of the furrow, tend mutants lack the SMW 2Corresponding author: Department of Molecular, Cell and Develop- mental Biology, 2204 Life Science, 621 Charles E. Young Dr. South, and, as a result, have a dramatically reduced number Los Angeles, CA 90095. E-mail: [email protected] of accessory cells. Importantly, in tend mutant cells, cell Genetics 174: 525–533 (September 2006) 526 T. S. Vivian Liao et al. Figure 1.—Crossing scheme for genetic screen. A total of 75,000 chromosomes were mu- tagenized and screened for the adult glossy-eye phenotype. A total of 90 glossy-eye mutants were isolated, and larval eye-disc clones were induced to study their BrdU incorporation and ELAV expression. Twenty-three failed to incorporate BrdU but expressed ELAV normally and were studied further. m*repre- sents the EMS-induced muta- tion. The cl-R3 mutation is cell lethal and is used to eliminate cells that are homozygous for the chromosome that does not contain m*. Similarly, for larval clones, cells homozygous for the RpS3 (Minute)mutation are eliminated. Cells heterozy- gous for the cl-R3 and RpS3 mutations grow more slowly, allowing m*/m* cells to form large clones. divisions in second and early third instar eye discs are chromosomes under the control of the eyeless enhancer, not affected. As a result, tend mutants give rise to large specifically in the embryonic anlagen of the developing clones in the adult eye. This is different from the eye (Newsome et al. 2000). The resulting clones of cells phenotype resulting from mutant clones in the basic will be homozygous for the newly induced mutation and cell cycle machinery, which would not be able to give rise are white due to the lack of the w1 gene essential for to any mutant clones. Furthermore, early patterning pigmentation. A total of 75,000 adult flies were screened and differentiation of neurons are also not affected in for the mosaic glossy-eye phenotype. Ninety mutants tend, as mutant clones express pan-neuronal, as well as were isolated. Complementation analysis was then per- cell-type-specific, markers and extend axons normally to formed by crossing each of the 90 mutants with one the optic lobe (Mandal et al. 2005). another and looking for lethality in the offspring. This On the basis of the nature of the tend phenotype, we yielded a total of 46 complementation groups. All hypothesized that a simple screen that identifies glossy- mutants were balanced over a TM6B Tb, y1 chromosome eye flies, followed by a secondary screen for a subset of and maintained as stocks. the mutants in which cells exhibit proliferation defects Next, third larval instar eye imaginal discs from the but have relatively normal patterning in the third larval identified glossy-eye mutants were assayed for BrdU instar, will potentially identify nuclear genes that en- incorporation and stained with the neuron-specific code mitochondrial proteins. Our hypothesis is sup- ELAV antibody (Yao et al. 1993). Larval eye-disc clones ported by our earlier finding that, like tend, flies mutant were generated by crossing the mutant stocks to yw in the mitochondrial components pdsw, mRpL4, and ey-flp; FRT82B Ubi-GFP RpS3/TM6B Tb, y1 flies. Clones mRpL17 exhibit a glossy-eye phenotype and fail to in- homozygous for the mutation are negatively marked corporate BrdU in the larval eye disc but stain normally by their lack of GFP. The 46 complementation groups with the ELAV antibody (Mandal et al. 2005). In this isolated from the glossy-eye screen fall into four catego- article, we show that an unbiased screen for glossy-eye ries on the basis of this characterization. Ten mutant mutants that exhibit these larval phenotypes is remark- groups exhibit both wild-type neuronal patterning and ably effective in identifying genes with mitochondrial BrdU incorporation, indicating that the glossy-eye phe- function. notype is due to mutations in genes that function after We conducted an eyeless-flp/FRT-based mitotic recom- the third instar and are required for cone and pigment bination screen (Newsome et al. 2000) on chromosome cell specification. Three mutant groups display abnor- arm 3R (Figure 1). y w ey-flp; FRT 82B males were mu- mal ELAV staining patterns while cells incorporate tagenized with 25 mm ethyl methanesulfonate (EMS) BrdU normally. Since the formation of cone cells re- and crossed to y w ey-flp; FRT82B P[w1] cl-R3/TM6B Tb, quires a combination of signals from differentiated y1 females to generate adult eye clones. ey-flp drives neuronal photoreceptors (Flores et al. 2000), this phe- mitotic recombination between the FRT-containing notype likely occurs because of defects in the patterning Note 527 TABLE 1 Mapping results and mutations in nuclear-encoded genes with mitochondrial function that exhibit a G1–S block in the third larval instar with normal neuronal differentiation A. Mapping results Cytological position Gene name Mitochondrial process Alleles B. Mitochondrial genes identified by the glossy eye–BrdU screen 84E4 CG10092 arginyl tRNA Protein biosynthesis–tRNA JR70, JR77, JV83, JR101, JR203, ligase (atl) synthesis JV219, JR247, JV250, JP782 84F11 CG9613 coenzyme Q biosynthesis Oxidative phosphorylation–complex III JV259, JP768 protein 2 (coq2) 91E4 glutamyl-tRNA amidotransferase Protein biosynthesis–tRNA synthesis JR15, JV87, JR94, JR113, JR205 A (gatA) 91F1 ATP synthase subunit d Oxidative phosphorylation–complex V JR92, JV115, JR238 (ATPsyn-d) of electron transport chain 94A1 CG6022 cytochrome c Oxidative phosphorylation–complex IV JV32, JV56, JV193, JR214; heme lyase (cchl) of electron transport chain Exelixis c04553 (Thibault et al. 2004) 94B6 mitochondrial ribosomal Protein biosynthesis–ribosome JV28 protein large subunit assembly 45 (mRpL45) Previously identified mitochondrial genes that cause the glossy-eye–defective BrdU incorporation phenotype 23F3 pdsw Oxidative phosphorylation–complex I pdswk10101 (Spradling of electron transport chain et al. 1999) 35F11 mitochondrial ribosomal protein large Protein biosynthesis–ribosome mRpL4k14608 (Spradling subunit 4 (mRpL4) assembly et al. 1999) 61B3 mitochondrial ribosomal protein large Protein biosynthesis–ribosome mRpL17KG06809 (Bloomington subunit 17 (mRpL17) assembly Drosophila Stock Center at Indiana University) 86F9 cytochrome c oxidase subunit Va (CoVa) Oxidative phosphorylation–complex IV JP785 (Mandal et al.
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